Loss of roost sites in buildings represents the major threat to Myotis myotis populations in the Czech Republic. To identify features that may determine roost selection by M. myotis, we compared a range of structural and habitat variables for 17 maternity roosts and 17 unoccupied, but potentially suitable, buildings in the Moravian Karst (Czech Republic). Roosts and control sites were mainly in churches and chateaus and all were detached from the surrounding buildings and uninsulated. The only difference between habitat surrounding roost and control buildings was that roost buildings had relatively lower amounts of hedges as linear connective features. Our results suggest that bats do not select building features from among suitable detached and uninsulated churches and chateaus and that bats tend to select building roosts that are not connected to woodland by hedges. Protection of roosts is an important conservation issue for female M. myotis and suitable roost sites such as detached and uninsulated buildings that are not connected to woodland by hedges are important maternity roosting resources.